TY - THES A1 - Schmidt, Selina T1 - Effects of biocides on processes underlying resistance evolution N2 - Antimicrobial resistance (AMR) is a global health problem. It is well known that antibiotics can drive evolutionary processes that underlie antimicrobial resistance (AMR) evolution and spread in clinical and environmental settings. In contrast, less is known about the effects of antimicrobial substances that are used as biocides (i.e. disinfectants and preservatives) on AMR evolution and spread. Biocides are present in various settings, interacting with diverse microbial communities. Therefore, it is crucial to evaluate their role in the evolution and dissemination of antimicrobial resistance. Biocides occur in a wide range of concentrations in various environmental settings. By examining how the various concentrations affect selection mechanisms, we gain insights into potential developments related to antimicrobial resistance. The aim of this PhD thesis is to investigate the effects of biocides on processes underlying resistance evolution. Specifically, the work focused on key mechanisms for resistance spread, resistance evolution, and the effect of selection pressures on evolved resistance mechanisms. The thesis is structured around three major objectives: (i) to determine the effect of biocides on the evolution of resistance by affecting the rate of occurrence of de novo mutations, (ii) to determine the effect of biocides on the spread of resistance genes by modifying the rate of horizontal gene transfer (HGT) processes, and (iii) to investigate the selective drivers of the emergence of antimicrobial resistance in adaptive laboratory evolution (ALE) experiments. De-novo mutations are spontaneous mutations that occur at a certain rate in microorganisms. The effect of biocides at subinhibitory environmentally relevant concentrations on the mutation rate in Acinetobacer baylyi, Bacillus subtilis and Escherichia coli was assessed with the fluctuation assay. The results showed that biocides affected mutation rates in a species and substance dependent matter. The bisbiguanide chlorhexidine digluconate, the quaternary ammonium compound didecyldimethylammonium chloride, the metal copper, the pyrethroid-insecticide permethrin, and the azole-fungicide propiconazole increase mutation rates in E. coli, whereas no increases were identified for B. subtilis and A. baylyi. Horizontal gene transfer refers to diverse mechanisms that mediate the transfer of mobile genetic elements between microorganisms. This work focused on conjugation and transformation. Conjugation is a process whereby a conjugative plasmid is transferred from a donor cell to a recipient cell. Transformation is a process whereby exogenous donor DNA is taken up into a recipient cell and integrated into the recipient’s’ genome. The effects of subinhibitory environmentally relevant biocide concentrations on the conjugation rate of E. coli and the transformation rate of the naturally competent organisms A. baylyi in were assessed. The results showed that benzalkonium chloride (BAC), chlorhexidine and permethrin increased conjugation in E. coli, while none of the biocides increased transformation rates in A. baylyi. To further understand the molecular mechanisms underlying the effects on mutation and conjugation rates, I investigated the induction of the RpoS-mediated general stress and the RecA-linked SOS response upon biocide exposure. The results show a link between the general stress and the SOS response with increased rates of mutation and conjugation, but not for all biocides. One major approach to study the evolutionary response of bacteria to antimicrobials are ALE experiments with growth at subinhibitory concentrations linked to serial subculturing over many generations. Such experiments have been used to study resistance evolution to antibiotics and biocides. However, previous work showed that adaptation to biocide stress may be mediated by different evolutionary drivers. Here, I investigated the contributions of evolution for increased survival as opposed to improved growth in ALE experiments with E. coli exposed to subinhibitory BAC concentrations. Two distinct evolutionary treatments selecting for survival only or survival and growth led to specific evolutionary adaptations apparent in the phenotypes and genotypes of the evolved populations. Populations growing in the presence of BAC evolved increased fitness in the presence of BAC associated with higher resistance to BAC and cross-resistance to antibiotics, while this was not the case for populations evolving for increased survival only. Genotypic characterization by whole genome sequencing of the evolved populations revealed parallelism in mutated genes among replicate populations and distinct differences across treatments. Treatments selecting for survival and growth showed mutations in stress response related genes (hslO and tufA), while selection for survival led to mutations in genes for metabolic regulation (cyaA) and cellular structure (flagella fliJ). In summary, this thesis shows that biocides affect AMR evolution and emphasizes the importance of understanding of how biocides impact the molecular and evolutionary process that underlie AMR evolution. KW - Biocides KW - Antimicrobial resistances KW - Microbial survival mechanisms PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:188-refubium-43383-9 SP - 1 EP - 101 PB - Freie Universität CY - Berlin AN - OPUS4-60678 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Braetz, S. A1 - Nordholt, Niclas A1 - Nerlich, A. A1 - Schreiber, Frank A1 - Tedin, K. A1 - Fulde, M. T1 - TisB enables antibiotic tolerance in Salmonella by preventing prophage induction through ATP depletion N2 - Antibiotic persistence comprises drug-tolerant bacteria that can survive treatment with antibacterial agents, despite lacking classical genetic resistance mechanisms. Therefore, persisters are clinically relevant because they can lead to treatment failures and chronic infections. Additionally, antibiotic persistence facilitates the evolution of resistance through genetic mutations. Persisters are triggered by a lack of nutrients, bacterial toxins, low ATP levels, or other stress responses that shut down bacterial metabolism. However, the involvement of prophages, viruses that integrate into bacterial chromosomes, is less well understood. In this study, we tested a tisAB deletion in Salmonella Typhimurium and examined persister cell formation following treatment with the DNA-damaging drug ciprofloxacin. TisB is a bacterial toxin that increases the influx of protons across the inner bacterial membrane into the cytosol, causing ATP depletion. We demonstrate that the deletion of tisAB increases prophage induction and bacterial killing, leading to a reduced persister cell fraction. The tisAB mutant is unable to down regulate its ATP concentration after exposure to ciprofloxacin, which in turn allows for stronger binding of RecA to single-stranded DNA, the activator of both the SOS response and prophage induction. KW - Antimicrobial resistance KW - Bacterial survival mechanisms KW - Escherichia coli KW - Salmonella typhimurium PY - 2025 DO - https://doi.org/10.1371/journal.ppat.1013498 IS - 9 SP - 1 EP - 23 AN - OPUS4-64642 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Identification of metabolic niches and their association to the persistence of antibiotic resistant bacteria in wastewater N2 - Wastewater treatment plants (WWTP) are hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that often arise with resistance. Recent research efforts are striving to uncover the role of bacterial metabolism for the ecology and evolution of antibiotic resistance. The aim of this study is to understand the ecological mechanisms, which allow ARB to persist in the environment. More specifically, we aim to identify metabolic niches that can select for and against resistant bacteria. 62 E. coli strains isolated from different WWTPs with different levels of resistance to 14 antibiotics and 3 disinfectants were assembled, sequenced, and phenotypically characterized. Next, genome scale metabolic models (GEMs) were constructed, and the growth of the strains was simulated in the presence of 298 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources to verify the model predictions. Competition experiments with synthetic microbial communities consisting of a selection of 10 WWTP isolates, 5 of which were antibiotic resistant and 5 sensitive, were carried out in minimal medium with different carbon sources. Population dynamics modelling was used to simulate the competition of isolates under different conditions. The isolates have a wide range of susceptibility to the antibiotics, while disinfectants result in a narrower range of susceptibility. GEMs identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-malate. A range of WWTP isolates were identified which use D-malate as carbon source and are susceptible to specific antibiotics. In contrast, antibiotic-resistant WWTP isolates were identified that did use sucrose as carbon source but not D-malate. Competition experiments demonstrated that changing the carbon source of the medium from sucrose to D-malate resulted in selection against the resistant isolates. Modelling the competition between isolates under different conditions suggests that adding a carbon source to a bacterial community under specific conditions could exclude resistant bacteria from a microbial community. Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally in complex microbial communities, it could be applied to reduce the number of ARB in environments such as wastewater. T2 - FEMS micro Konferenz CY - Milan, Italy DA - 14.07.2025 KW - AMR KW - Wastewater KW - Genome-scale metabolic model PY - 2025 AN - OPUS4-64659 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - A Customizable Procedure for Predicting Antibiotic Resistance Selection on Novel Antimicrobial Coatings N2 - The global burden of antimicrobial resistance (AR) has manifested in an increasing number of deaths attributable to antibiotic-resistant bacteria (ARB) in recent years, with projections indicating a continued rise. Combating AR requires a multifaceted approach, one aspect of which involves preventing the spread of ARB in hospital environments via high-touch surfaces, which are known contributors to nosocomial infections. In conjunction with routine disinfection protocols and infection prevention measures, antimicrobial surfaces or antimicrobial coatings (AC) are increasingly being investigated and implemented to reduce microbial transmission via high-touch surfaces, thereby mitigating their spread in healthcare settings. However, similar to antibiotics, prolonged use of such surfaces may lead to the direct or indirect selection of ARB. To prevent this, targeted tests must be developed to predict potential AR selection before AC implementation. Materials and Methods As part of the Horizon-Europe-funded project "STOP" (Grant Agreement ID: 101057961), a novel antimicrobial coating is being developed and tested to reduce pathogen transfer on surfaces without selecting for ARB. To this end, two bacterial libraries (80 strains each) were assembled from the two species most associated with AR-attributable mortality worldwide, Escherichia coli and Staphylococcus aureus, as identified in the most recent comprehensive study [1]. Each library maintains a 50/50% ratio of strains resistant or sensitive to third-generation cephalosporins (E. coli) and methicillin (S. aureus), respectively—representing two of the most widespread resistance profiles. An adapted ISO 22196 method was developed using a reference benchmark antimicrobial surface (i.e., copper), with stainless steel serving as a control. The goal was to infer potential advantages of copper-unrelated antibiotic resistance phenotypes on the tested surface. Results Metadata, genomic data, and antibiotic susceptibility testing (AST) data were collected for all selected strains in each library, ensuring diversity in sequence types (ST) of clinical relevance, antibiotic resistance gene profiles, and geographical origins. The method was calibrated on copper surfaces using a reference E. coli strain, establishing the initial parameters required for investigators to customize the test for a given AC. Procedural reproducibility was assessed by comparing results from independent operators. Discussion The two libraries are currently being screened against the selected copper surfaces to identify potential associations between the resistance phenotypes of the tested species and their observed survival rates post-exposure. Three mutually exclusive outcomes are anticipated: (i) a statistically significant survival advantage of resistant strains compared to sensitive strains, (ii) an inverse scenario where sensitive strains exhibit higher survival, or (iii) no significant difference between the two groups. Furthermore, post hoc principal component analysis utilizing metadata and AST data may help elucidate genetic traits that confer a survival advantage on the tested AC. Conclusions This test aims to assist developers of antimicrobial coatings and materials in assessing potential selective pressures toward ARB before these products are implemented and evaluated under real-life conditions. T2 - ESB Conference CY - Torino, Italy DA - 07.09.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64569 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Metabolic niches and persistence of antibiotic resistant bacteria in the environment N2 - Background and Aim: Wastewater treatment plants are considered as hotspots for the development and spread of antimicrobial resistance. Antimicrobial resistant bacteria (ARB) can persist in the environment for long periods of time, despite metabolic fitness costs that can arise with resistance. We are interested in understanding the mechanisms, which allow ARB to persist in the environment. More specifically, we want to identify metabolic niches that can select for resistant bacteria. Procedure/Method: 62 E. coli strains isolated from different WWTPs in Norway were used. The isolates have different levels of resistance to 14 antibiotics. The susceptibility of the isolates to 3 disinfectants was determined. In addition, genome scale metabolic models (GEMs) were constructed and the growth of the strains was simulated in the presence of 198 different carbon sources. Furthermore, the growth rates of the isolates were measured in the presence of 3 carbon sources. Findings/Results: Many of the isolates have high resistance to multiple antibiotics but only few of the isolates have higher minimum inhibitory concentrations to the disinfectants, compared to an E. coli laboratory strain. With the GEMs, we identified 40 carbon sources that can be utilized for growth only by a portion of all the isolates. The prediction accuracy of the GEMs was 93% in the case of D-Malate. A group of 10 isolates was identified of which 5 isolates are resistant to ciprofloxacin, gentamicin and tetracycline and can grow on Sucrose but not on D-Malate and the other 5 isolates are susceptible to the same 3 antibiotics but can grow on D-Malate and not on Sucrose. Implications/Applications: Our data suggest that changing the available carbon source could shift the selection advantage between resistant and susceptible bacterial strains. If this strategy is confirmed experimentally, it could be applied to reduce the number of ARB in environments such as wastewater. T2 - International Biodeterioration and Biodegradation Symposium 19 CY - Berlin, Germany DA - 09.09.2024 KW - Antimicrobial resistance KW - Wastewater PY - 2024 AN - OPUS4-61412 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - Antimicrobial coatings: modes of action, microbiological efficacy testing, and resistance evolution N2 - Within the Horizon-Europe funded project “STOP: Surface Transfer Of Pathogens”, Grant agreement 101057961, Working Package 6 is currently working on developing tests aimed at predicting possible selection and/or evolution of antibiotic resistance onto antimicrobial coatings meant for high-touch surfaces. This presentation describes the devised protocols and the preliminary results of their application on copper as a benchmark antimicrobial material. T2 - STOP-project Meeting CY - Sofia, Bulgaria DA - 27.02.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64567 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schreiber, Frank T1 - Mechanisms and evolution of resistance to antimicrobial biocides N2 - Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution, selection and transmission of AMR. These processes are impacted by pollution with antibiotics. However, antimicrobial biocides used as disinfectants and material preservatives are major pollutants exceeding the antibiotic market in terms of chemical diversity and mass. The aim of our work is to understand the mechanisms and risks of biocides for resistance and antibiotic cross-resistance evolution in bacteria to optimize their application and safeguard their efficacy. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. Importantly, widely used compounds such as chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations. Furthermore, we show that single-cell phenotypic heterogeneity regarding tolerance (persistence) determines survival against specific biocides including QACs and isopropanol. Mechanistic investigations reveal that known antibiotic persister mechanisms contribute to persister formation to biocides. The evolution of high-level tolerance to different biocides is linked to the initial persister level and the evolution of specific genetically encoded mechanisms related to properties of the cell envelope. Biocide-tolerant strains have a selective advantage in the presence of environmentally-relevant concentrations of antibiotics, which could lead to the stabilization of biocide tolerance in environments where biocides and antibiotics co-occur (e.g. wastewater, animal stables). Taken together, our work shows the importance of assessing the contribution of biocides on evolution and selection of AMR in the environment. T2 - EMBO Symposium on Mechanisms of drug resistance and tolerance in bacteria, fungi, and cancer CY - Heidelberg, Germany DA - 18.03.2025 KW - Antimicrobial surfaces KW - Biocides KW - Antimicrobial resistance PY - 2025 AN - OPUS4-64866 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - Antimicrobial coatings: modes of action, microbiological efficacy testing, and resistance evolution N2 - Antimicrobial coatings and materials on high touch surfaces can provide additional level of protection to periodic disinfection cycles by reducing bacterial loads. However, different coatings and materials assert their antimicrobial activity in different ways, and it is not clear whether they could pose detrimental selective pressure towards antibiotic-resistant bacteria. This talk elucidates modes of actions and current knowledge gaps in testing antimicrobial resistance on antimicrobial surfaces.” T2 - STOP Antimicrobial Coating Conference CY - Mons, Belgium DA - 05.12.2024 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64566 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Marano, Roberto T1 - A Customizable Procedure for Predicting Antibiotic Resistance Selection on Novel Antimicrobial Coatings N2 - This presentation introduceda new methodological approach developed to test antimocrobials implemented on high touch surfaces, inlcuding preliminary results on its use on reference antimicrobial materials. The seminar was organized by the 'Fast-real' project, funded by the Horizon Europe (project ID: 101159721). T2 - Novel strategies and considerations in fighting pathogens CY - Tartu, Estonia DA - 16.06.2025 KW - Antimicrobial-resistance KW - Surface antimicrobial testing KW - Materials testing PY - 2025 AN - OPUS4-64568 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Kanaris, Orestis T1 - Consequences of benzalkonium chloride tolerance on the development of antibiotic resistance in E. coli N2 - Biocides are used in large amounts in industrial, medical, and domestic settings. Benzalkonium chloride (BAC) is a commonly used biocide, for which previous research revealed that Escherichia coli can rapidly adapt to tolerate BAC-disinfection, with consequences for antibiotic susceptibility. However, the consequences of BAC-tolerance for selection dynamics and resistance evolution to antibiotics remain unknown. Here, we investigated the effect of BAC-tolerance in E. coli on its response upon challenge with different antibiotics. Competition assays showed that subinhibitory concentrations of ciprofloxacin - but not ampicillin, colistin and gentamicin - select for the BAC-tolerant strain over the BAC-sensitive ancestor at a minimal selective concentration of 0.0013-0.0022 µg∙mL-1. In contrast, the BAC-sensitive ancestor was more likely to evolve resistance to ciprofloxacin, colistin and gentamicin than the BAC-tolerant strain when adapted to higher concentrations of antibiotics in a serial transfer laboratory evolution experiment. The observed difference in the evolvability of resistance to ciprofloxacin was partly explained by an epistatic interaction between the mutations conferring BAC-tolerance and a knockout mutation in ompF encoding for the outer membrane porin F. Taken together, these findings suggest that BAC-tolerance can be stabilized in environments containing low concentrations of ciprofloxacin, while it also constrains evolutionary pathways towards antibiotic resistance. T2 - µClub Seminar CY - Berlin, Germany DA - 23.05.2025 KW - Biocides KW - AMR KW - Resistance evolution PY - 2025 AN - OPUS4-64658 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -